The present invention relates to quaternary ammonium functionalized dendrimers and methods of use therefor. Particularly, functionalized dendrimers comprised of Formula I are the object of the invention as well as their use as antimicrobial agents.
Jacobs and coworkers first described the antimicrobial activity of many quaternary ammonium compounds (QACs) in 1915. Jacobs, W. A.; Heidelberger, M. J. Biol. Chem. 1915, 20, 659-683, 685-694; Jacobs, W. A.; Heidelberger, M. J. Biol. Chem 1915, 21, 103-143, 145-152, 403-407, 439-453, 455-464, 465-475. The second, and most important event in the development of quaternary ammonium biocides occurred in 1935 when Domagk reported that the antibacterial activity of long-chain quaternary ammonium salts was significantly more potent than their short chain counterparts. Domagk, G. Deut. Med. Wochs., 61, 829 (1935). The markedly improved antimicrobial activity that occurred when a large aliphatic residue was attached to the quaternary nitrogen atom established the practicality and utility of these compounds, first in medicine, and later in many industrial applications. This important disclosure stimulated further research in synthesis and antimicrobial testing of QACs. It was shown that quaternary ammonium salts are most effective when one substituent is an alkyl chain with at least eight carbon atoms. Rahn, O.; Van Wseltine, W. Annual Review of Microbiology 1947, 1, 173. The issue of alkyl chain length was considered again when Cutler et al. studied how size affects the antimicrobial activity of a homologous series of alkyldimethylbenzyl ammonium chlorides. They found out that the highest potency is achieved when the alkyl chain has 14 carbons. Block, S. Disinfection, Sterilization and Preservation; 3rd ed.; Lea and Febiger: Philadelphia, 1983.
Quaternary ammonium compounds are currently widely used as disinfectants. They are surface-active, wide-spectrum antimicrobial agents. QACs are generally known to be more active against Gram-positive bacteria such as S. aureus than Gram-negative bacteria such as E. coli. 
Literally hundreds to thousands of polymeric compounds have been prepared and tested for antimicrobial properties. However, very few polymer compounds with biological activity have been discovered. The only commercially important polymeric biocide is biguanide. Biguanides show a wide spectrum of antimicrobial activity and are much more potent than the related monomers. Davies, A., Bentley, M. Field, B. S. J. Appl. Bacteriol., 31, 448-452 (1968).
Biofilms are matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces. Due to the destructive impact of biofilms in such diverse areas such as human physiology, food processing, and marine shipping, there is a great sense of urgency in the scientific community to discover chemical compositions and methods of use to solve biofilm-related problems.
Biofilms in human physiology can withstand host immune responses and are resistant to antibiotics. Antibiotics have proven to be ineffective in the prevention and treatment of biofihm infections particularly associated with biomaterial implants and prosthetic devices. Costerton, et al., Behaviour of Bacteria in Bioflims, ASM News, 55(12):650 (1989); AnWar, et al., Effective Use of Antibiotics in the Treatment of Biofilm-Associated Infections, ASM News, 58(12):665 (1992). Biofilm infections can occur either on dead/inanimate surfaces, such as sequestra of dead bone and medical devices, or on living tissues, as in the case of endocarditis. Biofilms grow very slowly; however, they are rarely resolved by any host defense system, even for those individuals with excellent immune systems. Bacterial colonization on implanted medical devices such as indwelling catheters, cardiac pacemakers, prosthetic heart valves, chronic ambulatory peritoneal dialysis catheters, and prosthetic joints, and the subsequent transformation into invasive infections contribute significantly to morbidity and complications related to implant-centered infections. Clinical studies have shown that even one thousand times the concentration of antibiotic that would typically kill planktonic (free-floating) bacteria fails to kill bacteria aggregated in the form of a biofilm. Costerton, et al., Annu. Rev. Microbiol., 49, 711-745 (1995); Costerton, J. W. J. Ind. Microbiol., 15, 137-140 (1995).
Biofilm bacteria are moreover resistant to bacteriophage and to a wide variety of antimicrobial agents used to combat biofouling in industrial environments.
Food processing, for example, can lead to food poisoning through such bacteria as, for example, Salmonella species, Clostridium perfringens, Bacillus species, Staphylococcus aureus, and Escherichia coli. Standard concentrations of various food processing industry standard disinfectants to clean surfaces in the industry is often ineffective thereby contributing to the public health risk factor. Walker, et al., Colloids and Surface A, 77, 225-229 (1993).
Biological fouling in the marine environment occurs on a variety of surfaces, including ships hulls, oil and gas installations and piers. Fouling on static structures increases loading forces caused by waves and currents on supports, and impedes inspection and maintenance. Biofilms in tubes and pipes can increase pressure drop and cause clogging. Biofouling on ships reduces their speed and maneuverability, due to the increased drag introduced by biological accumulations, causing increased fuel consumption and maintenance costs. Current marine anti-fouling technology employs biocides such as organotins which are blended into coatings as a preventive measure against growth of microorganisms. However, the ships treated in this manner require the arduous task of relatively frequent treatment. There is considerable interest in developing engineering approaches to modify susceptible surfaces to prevent biofilm formation. Several approaches have been actively investigated including modifying surfaces to minimize bacterial attachment or bacterial growth control through antimicrobial agents. Currently available approaches have not presented a formula for success. Moreover, traditional antibiological warfare agents are known to be very reactive and extremely toxic. Such agents include chlorine, formaldehyde, and peroxygen to destroy and or neutralize the effect of Anthrax spores and other biological entities. Accordingly, there also exists a need for antibiological warfare agents that are nonreactive and are virtually nontoxic to human skin.
Because of their compact structure and the availability of many end groups, dendrimers have attracted attention as possible antimicrobial agents. Zanini et al, Schengrund et al, and Bundle, et al. investigated carbohydrate modified dendrimers as antibacterial agents and using these oligosaccharides for treating and preventing bacterial and viral disease. Zanini, D.; Roy, R. J. Am. Chem. Soc., 119, 2088-2095 (1997); Roy, et al, J. Chem. Soc. Chem. Commun., 1869-1872 (1993); Schengrund et al, WO patent 9826662, 1998; U.S. Pat. No. 5,962,423, 1999. All these investigators took advantage of the multiple end groups of dendrimers and introduced different carbohydrates onto the dendrimers. These modified dendrimers tend to enhance the carbohydrate-protein binding interactions and can be potent inhibitors for bacterial and viral infections. The carbohydrate chemistry utilizes the specific interaction of a the carbohydrate with the bacteria. The use of these specific interactions limit the application of these carbohydrate dendrimers since different bacteria only respond to different carbohydrates. The quaternary ammonium functionalized dendrimers of the current invention use non-specific interactions with the bacteria and are therefore effective against a wide range of bacteria, spores, yeast, fungi, and mold. Balogh et al. synthesized dendrimer nanocomposites, dendrimers with inorganic silver or silver ions, and tested their antibacterial properties. Balogh, L. Proc. Am. Chem. Soc. Div. Colloi. and Surf. Chem., 54. (1999). For these dendrimer nanocomposites, the dendrimer itself does not have any antibacterial property. The activity comes from the silver/silver ions. In contrast, the quaternary ammonium functionalized dendrimers of the current invention derive antibacterial properties from the dendrimer itself. The dendrimers of the current invention are different from all previous investigations in that the surface groups of the dendrimers were transformed into quaternary ammonium groups. Unlike known QACs, the quaternary ammonium functionalized dendrimers of the current invention are more effective against Grain-negative bacteria such as E. coli. 
A quaternary ammonium functionalized dendrimer of Formula I is provided: 
wherein wherein D is a dendrimer; n is the generation number of the functionalized dendrimer; z is an integer less than or equal to 2(n+1); x is an anion; R is a linking group; Y is an alkyl group or aryl group; A is an alkyl group or aryl group, and B is an alkyl group or aryl group.
Methods of using the functionalized dendrimers of Formula I as effective broad spectrum antimicrobial agents in the healthcare as well as in various industrial applications are also provided.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All publications and patents referred to herein are incorporated by reference.
Controlling the growth of a microrganism as used herein is intended to encompass effecting diminished proliferation and/or lethal results to microorganisms including but not limited to bacteria, spores, yeast, fungi, mold and multicellular microorganisms. Microorganisms listed in Table I are particularly preferred. The functionalized dendrimers of the current invention are particularly intended to be employed in connection with each of the materials, tissues and sites recited in Table I. A spore as used herein refers to various small or minute primitive reproductive bodies, or resistant resting cells, typically unicellular. Also refers to the dustlike asexual reproductive bodies of fungi. A spore can for example corresponds to B. anthracis or anthrax.
Exposing a microorganism to a quaternary ammonium functionalized dendrimer of the present invention as used herein is intended to encompass subjecting a microorganism to sufficient proximity to a functionalized dendrimer of the present invention to cause an effect in the growth or proliferation of one or more microorganisms including but not limited to physical interaction between the microorganism and the functionalized dendrimer. This is intended to encompass exposure/interactions in solution as well as solid phase exposure/interaction, and liquid/solid phase exposure.
For example, liquid/solid phase exposure of the quaternary ammonium functionalized dendrimer of the present invention to one or more microorganims may involve using the quaternary ammonium functionalized dendrimer in a spray mixed with a suitable liquid carrier. Spray as used herein refers to the quaternary ammonium functionalized dendrimer of the present invention with or without a suitable carrier liquid applied as a liquid stream, fine vapor, mist, small drops, aerosol, or non-aersol. This spray can then be used to expose a microorganism to a quaternary ammonium functionalized dendrimer in liquid form. The spray could be used for example to control the growth of a microorganisms in or on clothing and surfaces.
The functionalized dendrimers are also intended for industrial as well as medical and home use applications including but not limited to elements of protective coatings such as paints, handwash formulations, means for use in ointments and related topical applications, cosmetics, cleaning and/or disinfectant/sanitation products, and sanitation of recreational water such as swimming pools and spas. The functionalized dendrimers are also intended to be used as a component in coating fibers and filters. The functionalized dendrimers are also effective against Anthrax spore. The dendrimer biocides of the present invention are nonreactive and are virtually nontoxic to human skin.
The functionalized dendrimers can also be immobilized on the surface of materials to create efficient antimicrobial environments in a wide variety of applications including garments for protective use as well as biomaterials and prosthetic devices for medical use. For example quaternary ammonium functionalized dendrimer of the present invention can be immobilized to polymers, glass, and metals. Polymers can be for example polyurethanes. Other examples include polystyrene, rubber, polyethylene, polypropylene, and engineering plastics. Immobilized on the surface is defined as attachment of functionalized dendrimers to a surface by covalent bonding, ionic interaction, coulombic interaction, hydrogen bonding, crosslinking (e.g., as crosslinked (cured) networks) or as interpenetrating networks, for example.
A method of controlling the growth of a microorganisms is herein provided comprising exposing a microorganism to a quaternary ammonium functionalized dendrimer of the present invention. Functionalized dendrimers of the present invention may be employed in solution (or initially in solution which may dessicate or carrier solvent may evaporate for example for coatings, paint and the like) at an effective concentration to control the growth of microorganisms. The functionalized dendrimers of the present invention may be employed at a wide variety of concentrations. Concentration of up to 100% may be used. Otherwise functionalized dendrimer of the present invention can be effective at concentrations from 1 ppm to concentration in excess of 10%. Effective minimum concentrations of functionalized dendrimers of the present invention against common bacteria is contemplated to be in concentrations 5 ppm to 1 to 2%. Preferred concentration is from 10 ppm to 200 ppm. More preferred is 20 ppm to 100 ppm. The concentration depending upon the role at hand (e.g., employment as a pre-surgical handwash disinfectant, coating for a biomaterial or otherwise prosthetic device for internal use, or element of an industrial-use biocidal coating). Elements for the formulations of functionalized dendrimers described herein are well known and are described, for example, in U.S. Pat. Nos. 6,030,632, 5,869,073, 6,022,551; 5,906,808; 5,776,430; 5,597,561; 5,244,666; and 5,164,107, each of which is herein incorporated by reference.
Table I is a partial list of infections. Quaternary ammonium functionalized dendrimers provided herein are contemplated to be effective against these and other bacterial species as well as spores, mold, fungi and other multicellular microorganisms.
Anthrax is a typical biological weapon. Novel quaternary ammonium functionalized dendrimers of the current invention can denature Anthrax spore, such as avirulent B. anthracis spore. The quaternary ammonium functionalized dendrimer biocides can be used as a denaturing spray or be impregnated in a soldiers uniform. Table II shows the denaturing effect of quaternary ammonium functionalized dendrimer biocides on Avirulent B. anthracis spore.
The margin of error is 5-8% for the % of viable spores. The concentration of the Avirulent B. anthracis spore was 107 spores per ml. The contact time was 1 hour. Functionalized dendrimers of the present invention are moreover contemplated to provide effective protection against biological warfare agents such as anthrax. Embodiments which employ functionalized dendrimers of the present invention accordingly include a denaturing spray or protective clothing impregnated with functionalized dendrimers as described herein. Effectiveness against anthrax spore is contemplated to be in concentrations from 0.1% to 20%. Preferred concentration is from 1% to 10%.
R as used herein refers to a linking group to link the quaternary ammonium group to the surface group of the Dendrimer (D). The linking group R can be either rigid or flexible. The linking group R can be for example an alkyl or aryl group. R may preferably selected from the group consisting essentially of xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NH-Phenyl-CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94(CH2)xe2x80x94, xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94(CH2)4xe2x80x94, xe2x80x94(CH2)5xe2x80x94, xe2x80x94(CH2)6xe2x80x94, xe2x80x94(CH2)7xe2x80x94, xe2x80x94(CH2)8xe2x80x94, xe2x80x94(CH2)9xe2x80x94, xe2x80x94(CH2)10xe2x80x94, xe2x80x94(CH2)11xe2x80x94, xe2x80x94(CH2)12xe2x80x94, xe2x80x94(CH2)13xe2x80x94, xe2x80x94(CH2)14xe2x80x94, xe2x80x94(CH2)15xe2x80x94, xe2x80x94(CH2)16xe2x80x94, xe2x80x94(CH2)17xe2x80x94, xe2x80x94(CH2)18xe2x80x94, xe2x80x94(CH2)19xe2x80x94, and xe2x80x94(CH2)20xe2x80x94 as well as similar physiochemical structures. The linking group is at least one carbon atom in length, and may be C1-20 straight, branched or cyclic alkyls, in which one or more of the carbons may be replaced with an O, S, or N.
Y, A, or B Alkyl group or aryl groups as used herein refers to chemical entities having less than 40 carbon atoms. Y, A, and/or B may also be chloromethyl.
Dendrimers are well defined, highly branched macromolecules that emanate from a central core. Commercially available dendrimers include polyamidoamine (PAMAM) dendrimers and polypropylene imine (PPI) dendrimers. Dendriditic architecture brings a very high number of functional groups in a compact space. Dendrimers in the present invention can for example be selected from the group consisting of polyamidoamine dendrimers, polylysine based dendrimers, polyethylene oxide based dendrimers, silicon based dendrimers, polyether based dendrimer and polypropylene imine dendrimers. A polylysine based dendrimers refers to a dendrimer in which the backbone or structure consists essentially of polylysine. A polyethylene oxide based dendrimers refers to a dendrimer in which the backbone or structure consists essentially of polyethylene oxide. A silicon based dendrimers refers to a dendrimer in which the backbone or structure consists essentially of silicon. A polyether based dendrimer refers to a dendrimer in which the backbone or structure consists essentially of polyether.
Architecturally similar to dendrimers, hyperbranched polymers can be prepared using a one-pot synthesis, so they are typically polydisperse, structurally imperfect, and better positioned for industrial applications. Hyperbranched polymers can be for example polyethylene oxide based hyperbranched polymers, polyglycerol based hyperbranched polymer and silicon based hyperbranched polymers. A polyethylene oxide based hyperbranched polymer refers to a hyperbranched polymer in which the backbone or structure consists essentially of polyethylene oxide. A polyglycerol based hyperbranched polymer refers to a hyperbranched polymer in which the backbone or structure consists essentially of polyglycerol. A silicon based hyperbranched polymer refers to a hyperbranched polymer in which the backbone or structure consists essentially of silicon. Commercially available Hyperbranched polymers include Polyols from Perstop Inc. which is an example of a hyperbranched polyols and hybrane(trademark) from DSM. Hybrane as used herein refers to the commercially available hybrane(trademark) from DSM.
D as used herein refers to dendrimers that are highly branched macromolecules that emanate from a central core as well as hyperbranched polymers. When used in relation to the letter D, the word dendrimer refers to both conventional dendrimers that emanate from a central core as well as hyperbranched polymers. D can be selected for example from the group consisting essentially of polyamidoamine, polylysine based dendrimers, polyethylene oxide based dendrimers, silicon based dendrimers, polypropylene imine dendrimers, polyether dendrimers, polyethylene oxide based hyperbranched polymers, polyglycerol based hyperbranched polymers, silicon based hyperbranched polymers, hyperbranched polyols and hybrane(trademark) from DSM.(Geleen, Netherlands).
N as used in Formula I is nitrogen.
Through a series of reaction and purification steps, dendrimers grow radially outwards. At different stages of the synthesis, dendrimers are identified by generations. As the generation increases, the number of surface groups, the size of the dendrimer, and the molecular weight of the dendrimer increase.
The variable xe2x80x9cnxe2x80x9d as used herein refers to the generation of the Dendrimer D. Therefore D1, D2 or D3 represent dendrimers of generation 1, 2, and 3 respectively
The variable xe2x80x9czxe2x80x9d as used herein refers to the number of surface functional groups for a dendrimer. As the generation, n, of any dendrimer increases, the number of surface functional groups, z, increases. A generation 1 dendrimer, D1, may have as many as many as four surface functional groups, while a generation 2 dendrimer, D2, may have a many as eight surface functional groups. The maximum number of functional groups is represented by the letter z when it is equal to 2n+1 where n is equal to generation of a given dendrimer. Because a given dendrimer may have less than the maximum number of possible surface groups functionalized, the actual number of functionalized groups on a given dendrimer is less than or equal to 2n+1.
The variable xe2x80x9cxxe2x80x9d as used herein refers to an anion. The variable element of the invention x is preferably selected from, but not limited to chloride, bromide, sulfate, nitrate, chlorate, tetrafluoroborate, perchlorate, hexafluorophosphate, permanganate, sulfite.
The advent of dendrimers represents a major breakthrough in synthetic chemistry. Dendrimers can be tailored to generate uniform or discrete functionalities and possess tunable inner cavities, surface moieties, sizes, molecular weights, and solvent interactions. Dendrimers can be synthesized by a convergent approach. Tomalia, et al. Macromolecules, 20, 1164. (1987). Dendrimers can also be synthesized by a divergent approach. Tang, et al. Bioconjugate Chem., 7, 703-714. (1996). In the divergent approach, growth of dendrimers dendrimers starts from a multi-functional core. Through a series of reaction and purification steps, dendrimers grow radially outwards. At different stages of the synthesis, dendrimers are identified by generations. As the generation increases, the number of functional groups, the size of the dendrimer, and the molecular weight of the dendrimer increase. At a certain stage of the synthesis, steric hindrance prevents one from achieving the, where the highest generation is synthesized. Commercially available dendrimers, such as polyamidoamine (PAAM) dendrimers from Dendritech Inc. (Midland, Mich., USA) and polypropylene imine (PPI) dendrimers from DSM (Geleen, Netherlands) are synthesized by the divergent approach. In the convergent approach, dendrons, as parts of dendrimers, are synthesized according to the divergent approach and these dendrons are then coupled to a multifunctional core. The advantage of the convergent approach is that the chemistry of each dendron can be different, and distinct functional groups can be integrated into dendrimers at precise sites. Due to the repetitive nature of the dendrimer synthesis and the extensive purification required to achieve the, dendrimers are very expensive and not readily available. The combination of discrete numbers of functionalities in one molecule and high local densities of active groups has attracted a lot of attention, especially for biological applications. The unique architecture of dendrimers, they have been investigated for a wide variety of applications, such as gene delivery vesicles, Tang, et al., Bioconjugate Chem. 7, 703-714 (1996); Kukowska-Latallo, et al., Proc. Natl. Acad. Sci. USA, 93, 4897-4902 (1996), catalysts, Zeng, F. Z., S. C. Chem. Rev., 97, 1681 (1997); Newkome, et al., Chem. Rev., 99, 1689-1746 (1999), drug delivery carriers, Liu, M.; Frechet, J. M. J. Proc. Am. Chem. Soc. Polym. Mater. Sci. Engr., 80, 167 (1999); Uhrich, K. TRIP, 5, 388-393 (1997); Liu, H.; Uhrich, K. Proc. Am. Chem. Soc. Div. Polym. Chem., 38, 1226 (1997), chromatography stationary phases, Matthews, et al., Prog. Polym. Sci., 23, 1-56 (1998), boron neutron capture therapy agents, Newkome, et al., Dendritic Macromolecules: Concepts, Syntheses, Perspectives; VCH: Weinheim, Germany, (1996); Newkome, G. R. Advances in Dendritic Macromolecules; JAI Press: Greenwich, Conn., Vol. 2. (1995), and magnetic resonance imaging contrast agents. Tomalia, D. A. Adv. Mater., 6, 529-539. (1994).
Dendrimers can offer a high local concentration of functional groups. A functionalized dendrimer is a dendrimer with surface groups that have been replaced with a chemical functional group. A surface groups is the chemical groups at the terminal ends of the branches or backbone of a dendrimer. Surface groups can be for example xe2x80x94NH2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94CN. For example a polypropylene imine (PPI) dendrimer is terminated with xe2x80x94NH2 surface groups. Chemical groups can be added to these surface groups such the resulting dendrimer is terminated by a xe2x80x94NHxe2x80x94W group wherein W is some functional chemical group. Functionalized dendrimers with biologically active groups, results in an increased potency associated with the high local concentration. Once a dendrimer has been functionalized, they may be called functionalized dendrimers or modified dendrimers. The resulting functionalized dendrimer can be represented by Dn-(W)z. The dendrimer can be referred to through use of xe2x80x9cDxe2x80x9d to refer to the dendrimer structure except for the chemical functional group represented by W. The generation of the dendrimer D can be represented by the letter n. The letter xe2x80x9cnxe2x80x9d a defined herein represents the generation of the Dendrimer D. Therefore D1, D2 or D3 represent dendrimers of generation 1, 2, and 3 respectively. As the generation of any dendrimer increases, the number of potential surface functional groups, z, increases. A generation 1 dendrimer, D1, may have as many as four surface functional groups, while a generation 2 dendrimer, D2, may have a many as eight surface functional groups. The maximum number of functional groups, is represented by the letter z, when it is equal to 2n+1 where n is equal to generation of a given dendrimer. Because a given dendrimer may have less than the maximum number of possible surface groups functionalized, the actual number of functionalized groups on a given dendrimer is less than or equal to 2n+1. During formation of the dendrimer biocide system, precipitation may be observed during the reaction, resulting in incomplete reaction and inhomogeneity of the dendrimer biocides. This results in the actual number z being variable across dendrimer biocides.
Quaternary ammonium compounds are currently widely used as disinfectants. A quaternary ammonium functionalized dendrimers is a functionalized dendrimer can be represented by Dn-(W)z wherein the chemical structure of the chemical group W is terminated by a quaternary ammonium compound.
Generally speaking, biocides immobilized on dendrimers can be more effective if the target sites are cell walls and/or cell membranes. Since there are many QACs on the dendrimer, the resulting dendrimer biocides are polycationic. Polycationic structure has been proven to improve the permeability of the bacterial membrane and facilitate the antimicrobial action of biocides. Antimicrobial functionalities may be placed onto dendrimers and used to combat microbial fouling/infections. Another complementary approach is to prevent the initial attachment of bacteria to surfaces. The attachment is usually a prerequisite for colonization of bacteria and invasion of tissues.
Quaternary ammonium functionalized dendrimers provided herein outperform known polymeric and small molecule biocides. The dendrimer architecture has been shown to be over 100 times more potent against E. coli as compared to their small molecule counterparts such as n-dodcecyltrimethyl ammonium chloride (DTAC). Small molecule QACs are not very effective on Gram-negative bacteria, such as E. coli, because these cells have very sophisticated outer membrane structures that effectively keep out antibacterial agents. However. with the combination of the high functional group density and the increased permeability due to the polycationic structure, dendrimer reach and disrupt cell membranes, eventually leading to cell death. Functionalized dendrimers comprised of Formula I can be used in any of a myriad of applications that requires a potent biocide/antimicrobial agent. Particularly the present invention is drawn toward quaternary ammonium functionalized dendrimers comprised of Formula I which effect a very high number of functional groups in a compact space and therefore increase local concentration of biocidal agents.
Quaternary ammonium functionalized dendrimers of the Formula I are subject of the current invention. 
A quaternary ammonium functionalized dendrimer of Formula I is provided: wherein D is a dendrimer; n is the generation number of the functionalized dendrimer; z is an integer less than or equal to 2(n+1); x is an anion; R is a linking group; Y is an alkyl group or aryl group; A is an alkyl group or aryl group, and B is an alkyl group or aryl group. Alkyls can be linear or branched. The groups A, B, and Y can all be the same or may all be different. Quaternary ammonium functionalized dendrimer according are preferred wherein D is selected from the group consisting essentially of polyamidoamine, polylysine based dendrimers, polyethylene oxide based dendrimers, silicon based dendrimers, polypropylene imine dendrimers, polyether dendrimers, polyethylene oxide based hyperbranched polymers, polyglycerol based hyperbranched polymers and silicon based hyperbranched polymers. Dendrimers of the current invention are preferred wherein n ranges from 1 to 15, 1-6 being most preferred. x represents an anion. The variable element of the invention x is preferably selected from, but not limited to chloride, bromide, sulfate, nitrate, chlorate, tetrafluoroborate, perchlorate, hexafluorophosphate, permanganate, sulfite. Linking group R may preferably selected from the group consisting essentially of xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94COxe2x80x94NH-Phenyl-CH2xe2x80x94, xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94(CH2)xe2x80x94, xe2x80x94(CH2)2xe2x80x94, xe2x80x94(CH2)3xe2x80x94, xe2x80x94(CH2)4xe2x80x94, xe2x80x94(CH2)5xe2x80x94, xe2x80x94(CH2)6xe2x80x94, xe2x80x94(CH2)7xe2x80x94, xe2x80x94(CH2)8xe2x80x94, xe2x80x94(CH2)9xe2x80x94, (CH2)10xe2x80x94, xe2x80x94(CH2)11xe2x80x94, xe2x80x94(CH2)12xe2x80x94, xe2x80x94(CH2)13xe2x80x94, xe2x80x94(CH2)14xe2x80x94, xe2x80x94(CH2)15xe2x80x94, xe2x80x94(CH2)16xe2x80x94, xe2x80x94(CH2)17xe2x80x94, xe2x80x94(CH2)18xe2x80x94, xe2x80x94(CH2)19xe2x80x94, and xe2x80x94(CH2)20xe2x80x94, as well as similar physiochemical structures. Y is preferably an alkyl or aryl group consisting of within the range of 1 to 32 carbon atoms. Y may be linear or branched. Y may be an alkyl group consisting of within the range of 1 to 32 carbon atoms. Y is preferably a linear alkyl group consisting of within the range of 8 to 24 carbon atoms. Y may be an aryl group selected-from the group consisting essentially of phenyl, xe2x80x94(CH2)-phenyl, xe2x80x94(CH2)2-phenyl, xe2x80x94(CH2)3-phenyl, xe2x80x94(CH2)4-phenyl, xe2x80x94(CH2)5-phenyl, (CH2)6-phenyl, xe2x80x94(CH2)7-phenyl, xe2x80x94(CH2)8-phenyl, xe2x80x94(CH2)9-phenyl, (CH2)10-phenyl, xe2x80x94(CH2)11-phenyl, xe2x80x94(CH2)12-phenyl, xe2x80x94(CH2)13-phenyl, xe2x80x94(CH2)14-phenyl, xe2x80x94(CH2)15-phenyl, xe2x80x94(CH2)16-phenyl, xe2x80x94(CH2)17-phenyl, xe2x80x94(CH2)18-phenyl, xe2x80x94(CH2)19-phenyl, xe2x80x94(CH2)20-phenyl, xe2x80x94(CH2)21-phenyl, xe2x80x94(CH2)22-phenyl, xe2x80x94(CH2)23-phenyl, and xe2x80x94(CH2)24-phenyl. Y may also be chloromethyl. A and B are alkyl or aryl groups, the same or different, each consisting of within the range of 1 to 32 carbon atoms. A and B can be liner or branched. A and B are preferably alkyl groups, the same or different, each consisting of within the range of 1 to 24 carbon atoms. A and B are most preferably linear alkyl groups, the same or different, each consisting of within the range of 1 to 24 carbon atoms; however, A and/or B may be an aryl group selected from the group consisting essentially of phenyl, xe2x80x94(CH2)-phenyl, xe2x80x94(CH2)2-phenyl, xe2x80x94(CH2)3-phenyl, xe2x80x94(CH2)4-phenyl, xe2x80x94(CH2)5-phenyl, xe2x80x94(CH2)6-phenyl, xe2x80x94(CH2)7phenyl, xe2x80x94(CH2)8-phenyl, xe2x80x94(CH2)9-phenyl, (CH2)10-phenyl, xe2x80x94(CH2)11-phenyl, xe2x80x94(CH2)12-phenyl, xe2x80x94(CH2)13-phenyl, xe2x80x94(CH2)14-phenyl, xe2x80x94(CH2)15-phenyl, xe2x80x94(CH2)16-phenyl, xe2x80x94(CH2)17-phenyl, xe2x80x94(CH2)18-phenyl, xe2x80x94(CH2)19-phenyl, xe2x80x94(CH2)20-phenyl, xe2x80x94(CH2)21-phenyl, xe2x80x94(CH2)22-phenyl, xe2x80x94(CH2)23-phenyl, and xe2x80x94(CH2)24-phenyl. A and/or B may also be chloromethyl. Quaternary ammonium functionalized dendrimers of Formula I is preferred wherein D is an NH2 terminated polypropylene imine (PPI) dendrimer and n is from generation 1 to 6 and wherein R is xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94 and x is Chloride; or R is xe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94 and x is Bromide. Quaternary ammonium functionalized dendrimer are preferred wherein Y is preferably a linear alkyl group consisting of within the range of 8 to 24 carbon atoms. Functionalized dendrimers of the invention are particularly preferred wherein A and B are xe2x80x94CH3.
Quaternary ammonium compounds are usually synthesized by the addition of an alkyl halide to a tertiary amine. Davis, B., Recent Developments in the Technology of Surfactants; Porter, M. R., Ed.; Elservier Applied Sciences: London, ppp 70 (1990). This reaction is reversible at high temperature, so 30-80xc2x0 C. is typically used for the synthesis. The quaternary ammonium salts are nearly insoluble in diethyl ether and benzene, sparingly soluble in acetone, and freely soluble in water and alcohol.
Five generations, for example, of poly(propylene imine) (PPI) dendrimers , D, with 4-64 primary amine surface groups, z, have been used to synthesize dendritic biocides. The functionalization of dendrimers consists of two steps. The first involves introducing halogen, such as chlorine or bromine, functionality to the dendrimer by reacting primary amine groups of the dendrimer with a bifunctional chemical such as 2-chloroethyl isocyanate or 2-bromoethyl isocyanate. The halogen can then react with tertiary amines to form quaternary ammonium compounds and with phosphines to form quaternary phosphonium salts. A homologous series of tertiary amines with the alkyl chain ranging from C8 to C16. Five times excess of the amine is typically used to facilitate the reaction and to prevent intra-dendrimer quaternization. The final products are usually obtained as yellow powders. For dendrimer biocides that were used in polyurethane functionalization, the chemistry is similar. However, the stoichiometry was adjusted so that there were still some free pendent halide groups left for the immobilization chemistry. In practice, usually only 80-90% of stoichiometric amount of tertiary amine is used.
For the reaction of the primary amine terminated dendrimer with 2-bromoethyl or 2-chloroethyl isocyanate, the reaction itself is very fast and can finish in seconds. Since the reaction is exothermic, cross-linking can happen if the heat is not properly removed. Therefore, the isocyanate may be added dropwise into the dendrimer solution. An ice bath may be used to lower the reaction temperature. There is not any limit in choosing solvents as long as the isocyanate-modified dendrimers are soluble in the solvent.
The reaction of a tertiary amine and the modified dendrimer finally results in a dendrimer biocide. The reaction rate can be enhanced in polar solvents such as DMF. Elevated temperature also improves the reaction rate; however, high temperature also leads to the reverse reaction, the degradation of quaternary ammonium compounds. The reaction can be conducted in several solvents. The reaction rate is in the order of DMF greater than 1-butanol greater than acetone. Sommer and co-workers also found similar rate dependence on solvent and that the relative quaternization rate in various solvents is 900:285:70:1 (DMF:methanol:butanol:hexane). Sommeretal., J. Org. Chem., 26, 824-828 (1971).
Compared to conventional polymers, the end groups of dendrimers play a more important role in determining their solubility. For example, the hydrophobically modified dendrimers, no matter what the interior structure of the dendrimers is, are soluble in hydrocarbons such as hexane and toluene. The solubility is primarily determined by the alkyl chain on the surface. Therefore, surface modification can lead to a significant difference in the solubility of the dendrimers. An appropriate solvent or solvent mixture must be selected if complete modification of the dendrimers is desirable. For the dendrimer biocide system, if DMF is used alone, precipitation may be observed during the reaction, resulting in incomplete reaction and inhomogeneity of the dendrimer biocides. Since the dendrimer gets more and more hydrophobic with the process of the reaction, addition of toluene to accommodate the hydrophobic part is required. Typically a 2:1 ratio of DMF:toluene mixture may be used for the quaternization reaction. The ratio can only be determined empirically and depends on the amount of the tertiary amine used in the reaction.
After the reaction, quaternary ammonium salts are repeatedly precipitated in large volumes of acetone. These precipitates are extremely hygroscopic. The precipitates is filtered, redissolved in a minimal amount of ethanol, and repeatedly stripped with anhydrous toluene to remove moisture. The samples are dried for xcx9c120 hr at 65xc2x0 C. in a vacuum oven and stored in a vacuum desiccator. Some samples appeared to be crystalline while others were amorphous. For the amorphous samples, recrystallization in acetone:methanol (9:1, v/v) improved sample purity.
These dendrimer biocides have a strong tendency to trap some excess tertiary amine and solvent. To further purify the samples, dialysis or diafiltration with a membrane with a 1000-2000 cut-off molecular weight is required. The dialysis process is typically very slow. A semi-continuous process called diafiltration, a combination of dialysis and ultrafiltration, may be used for purification. The diafiltration usually takes 2-3 days. The diafiltration may be stopped when the exit stream does not contain any tertiary amine or other small molecules detectable by a gas chromatography-mass spectrometer (GC-MS). The dendrimer biocides are also highly hygroscopic. Therefore, storage in vacuum desiccator is required.
The versatile chemistry allows for the preparation of a series of dendrimer biocides with different hydrophobes by using different tertiary amines, with different molecular weight, size and number of functional groups by using different generations of dendrimers, and with different counter-anions by using different isocyanates such as 2-bromoethyl isocyanate. These dendrimerbiocides are soluble in alcohol, chloroform, and dimethylformamide; slightly soluble in water; and not soluble in tetrahydrofuran, toluene, and acetone.
Table III represents potential dendrimers based on poly(propylene imine) (PPI) dendrimers. For the purposes of the following discussion and table a shorthand of DnXNY based on Formula 1 is used. Where D is a poly(propylene imine) (PPI) dendrimers if generation n. Having an anion X and Y group. When using the preceding nomenclature, groups A and B of formula are both xe2x80x94CH3, and the linking group, R, is represented by xe2x80x94NHxe2x80x94CONHCH2CH2xe2x80x94 when the anion is chloride (Cl) and xe2x80x94NHxe2x80x94CONHCH2xe2x80x94 when the anion is Bromide (Br). Therefore D1ClNC12 is a generation 1 poly(propylene imine) (PPI) with a chloride anion and Y group represented by an alkyl C12.
The antibacterial activity of the dendrimer biocides depends on the generation, n, (size, molecular weight) of the dendrimer, the chain length of the hydrophobe (A, B, and Y) on the quaternary ammonium ion and the counter-anion X. The biocidal activity is in the order of D5ClNC12 greater than D4ClNC12 greater than D1ClNC12 greater than D2ClNC12 greater than D3ClNC12.
High charge density also plays a substantial role in determining antimicrobial properties of polycationic biocides. Since the active species are the cations, different counter-anions can affect antimicrobial properties by influencing disassociation of polycationic biocides in water. D3BrNC14 is more potent than D3ClNC14. While D3ClNC14 is not so effective in killing E. coli at 1.8 mM, D3BrNC14 reduces the relative bioluminescence of the bacteria to about 0.2% in 1 hour. The potency difference of dendrimer biocides with chloride and bromide counter-anions was not expected since both these ions tend to dissociate freely in water.
To verify that the antimicrobial effect of the dendrimer biocides is not bacteria dependent, their antibacterial properties against Gram-positive bacteria Staphylococcus aureus were investigated by suspension tests. Results show that D4ClNC12 inhibited the growth of S. aureus as low as 1 ppm and effectively killed them at 10 ppm in 60 min. Somewhat less effective results have been reported for DTAC. This demonstrates the strong potency of dendritic biocides on typical gram-positive bacteria S. aureus.